S-Nitrosoglutathione (GSNO) has been used as a nitric oxide donor compound and has also been postulated to be involved in the transport of nitric oxide in vivo. In this study we have examined the possibility that GSNO is a substrate for gamma-glutamyl transpeptidase (gamma-GT), an enzyme that hydrolyses the gamma-glutamyl moiety of glutathione to give glutamate and cysteinylglycine. gamma-GT accelerated the decomposition of GSNO, forming S-nitrosocysteinylglycine (CG-SNO) by a mechanism inhibitable by the gamma-GT inhibitors acivicin and S-methylglutathione. The Km of gamma-GT for GSNO was found to be 28 micromole. In the presence of contaminating transition metal ions, gamma-GT accelerated the release of nitric oxide from GSNO, as CG-SNO is more susceptible to transition metal ion-dependent decomposition than GSNO. However, in the presence of the transition metal ion chelator diethylenetriaminepentaacetic acid, neither GSNO nor CG-SNO decomposed to generate nitric oxide. Neither S-methylglutathione nore acivicin affected the vasodilatory response to GSNO in an isolated perfused rat heart. However, rat kidney homogenate stimulated the decomposition of GSNO by an acivicin-inhibitable mechanism. It is likely therefore that gamma-GT is involved in the decomposition of GSNO in the kidney but not in the heart.